Angela Siciliano

Resveratrol accelerates erythroid maturation by activation of FOXO3 and ameliorates anemia in beta-thalassemic mice

Resveratrol, a polyphenolic-stilbene, has received increased attention in the last decade due to its wide range of biological activities. Beta(β)-thalassemias are inherited red cell disorders, found worldwide, characterized by ineffective erythropoiesis and red cell oxidative damage with reduced survival. We evaluated the effects of low-dose-resveratrol (5 μM) on in vitro human erythroid differentiation of CD34+ from normal and β-thalassemic subjects. We found that resveratrol induces accelerated erythroid-maturation, resulting in the reduction of colony-forming units of erythroid cells and increased intermediate and late erythroblasts. In sorted colony-forming units of erythroid cells resveratrol activates Forkhead-box-class-O3, decreases Akt activity and up-regulates anti-oxidant enzymes as catalase. In an in vivo murine model for β-thalassemia, resveratrol (2.4 mg/kg) reduces ineffective erythropoiesis, increases hemoglobin levels, reduces reticulocyte count and ameliorates red cell survival. In both wild-type and β-thalassemic mice, resveratrol up-regulates scavenging enzymes such as catalase and peroxiredoxin-2 through Forkhead-box-class-O3 activation. These data indicate that resveratrol inhibits Akt resulting in FoxO3 activation with upregulation of cytoprotective systems enabling the pathological erythroid precursors to resist the oxidative damage and continue to differentiate. Our data suggest that the dual effect of resveratrol on erythropoiesis through activation of FoxO3 transcriptional factor combined with the amelioration of oxidative stress in circulating red cells may be considered as a potential novel therapeutic strategy in treating β-thalassemia.

Erythrocyte membrane changes of chorea-acanthocytosis are the result of altered Lyn kinase activity

Acanthocytic RBCs are a peculiar diagnostic feature of chorea-acanthocytosis (ChAc), a rare autosomal recessive neurodegenerative disorder. Although recent years have witnessed some progress in the molecular characterization of ChAc, the mechanism(s) responsible for generation of acanthocytes in ChAc is largely unknown. As the membrane protein composition of ChAc RBCs is similar to that of normal RBCs, we evaluated the tyrosine (Tyr)-phosphorylation profile of RBCs using comparative proteomics. Increased Tyr phosphorylation state of several membrane proteins, including band 3, β-spectrin, and adducin, was noted in ChAc RBCs. In particular, band 3 was highly phosphorylated on the Tyr-904 residue, a functional target of Lyn, but not on Tyr-8, a functional target of Syk. In ChAc RBCs, band 3 Tyr phosphorylation by Lyn was independent of the canonical Syk-mediated pathway. The ChAc-associated alterations in RBC membrane protein organization appear to be the result of increased Tyr phosphorylation leading to altered linkage of band 3 to the junctional complexes involved in anchoring the membrane to the cytoskeleton as supported by coimmunoprecipitation of β-adducin with band 3 only in ChAc RBC-membrane treated with the Lyn-inhibitor PP2. We propose this altered association between membrane skeleton and membrane proteins as novel mechanism in the generation of acanthocytes in ChAc.

Peroxiredoxin-2 expression is increased in (β-thalassemic mouse red cells but is displaced from the membrane as a marker of oxidative stress

Peroxiredoxin 2 (Prx2), the third most abundant cytoplasmic protein in red blood cells (RBCs), is involved in the defense against oxidative stress. Although much is known about Prx2 in healthy RBCs, its role in pathological RBCs remains largely unexplored. Here, we show that the expression and net content of Prx2 are markedly increased in RBCs from two mouse models of beta-thalassemia (beta-thal; Hbb(th/th) and Hbb(th3/+) strains). We also demonstrate that the increased expression of Prx2 correlates with the severity of the disease and that the amount of Prx2 bound to the membrane is markedly reduced in beta-thal mouse RBCs. To explore the impact of oxidative stress on Prx2 membrane association, we examined Prx2 dimerization and membrane translocation in murine RBCs exposed to various oxidants (phenylhydrazine, PHZ; diamide; H(2)O(2)). PHZ-treated RBCs, which mimic the membrane damage in beta-thal RBCs, exhibited a kinetic correlation among Prx2 membrane displacement, intracellular methemoglobin levels, and hemichrome membrane association, suggesting the possible masking of Prx2 docking sites by membrane-bound hemichromes, providing a possible mechanism for the accumulation of oxidized/dimerized Prx2 in the cytoplasm and the increased membrane damage in beta-thal RBCs. Thus, reduced access of Prx2 to the membrane in beta-thal RBCs represents a new factor that could contribute to the oxidative damage characterizing the pathology.

Membrane association of peroxiredoxin-2 in red cells is mediated by the N-terminal cytoplasmic domain of band 3

Band 3 (B3), the anion transporter, is an integral membrane protein that plays a key structural role by anchoring the plasma membrane to the spectrin-based membrane skeleton in the red cell. In addition, it also plays a critical role in the assembly of glycolytic enzymes to regulate red cell metabolism. However, its ability to recruit proteins that can prevent membrane oxidation has not been previously explored. In this study, using a variety of experimental approaches including cross-linking studies, fluorescence and dichroic measurements, surface plasmon resonance analysis, and proteolytic digestion assays, we document that the antioxidant protein peroxiredoxin-2 (PRDX2), the third most abundant cytoplasmic protein in RBCs, interacts with the cytoplasmic domain of B3. The surface electrostatic potential analysis and stoichiometry measurements revealed that the N-terminal peptide of B3 is involved in the interaction. PRDX2 underwent a conformational change upon its binding to B3 without losing its peroxidase activity. Hemichrome formation induced by phenylhydrazine of RBCs prevented membrane association of PRDX2, implying overlapping binding sites. Documentation of the absence of binding of PRDX2 to B3 Neapolis red cell membranes, in which the initial N-terminal 11 amino acids are deleted, enabled us to conclude that PRDX2 binds to the N-terminal cytoplasmic domain of B3 and that the first 11 amino acids of this domain are crucial for PRDX2 membrane association in intact RBCs. These findings imply yet another important role for B3 in regulating red cell membrane function.

Deoxygenation affects tyrosine phosphoproteome of red cell membrane from patients with sickle cell disease

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder related to the production of a defective form of hemoglobin, hemoglobin S (HbS). One of the hallmarks of SCD is the presence of dense, dehydrate highly adhesive sickle red blood cells (RBCs) that result from persistent membrane damage associated with HbS polymerization, abnormal activation of membrane cation transports and generation of distorted and rigid red cells with membrane perturbation and cytoskeleton dysfunction. Although modulation of phosphorylation state of the proteins from membrane and cytoskeleton networks has been proposed to participate in red cell homeostasis, much still remains to be investigated in normal and diseased red cells. Here, we report that tyrosine (Tyr-) phosphoproteome of sickle red cells was different from normal controls and was affected by deoxygenation. We found proteins, p55 and band 4.1, from the junctional complex, differently Tyr-phosphorylated in SCD RBCs compared to normal RBCs under normoxia and modulated by deoxygenation, while band 4.2 was similarly Tyr-phosphorylated in both conditions. In SCD RBCs we identified the phosphopeptides for protein 4.1R located in the protein FERM domain (Tyr-13) and for alpha-spectrin located near or in a linker region (Tyr-422 and Tyr-1498) involving protein areas crucial for their functions in the context of red cell membrane properties, suggesting that Tyr-phosphorylation may be part of the events involved in maintaining membrane mechanical stability in SCD red cells.

Abnormal modulation of cell protective systems in response to ischemic/reperfusion injury is important in the development of mouse sickle cell hepatopathy.

Background Sickle cell disease, a genetic red cell disorder inherited in an autosomal recessive manner, occurs throughout the world. Hepatic dysfunction and liver damage may be present in sickle cell disease, but the pathogenesis of these conditions is only partially understood. Design and Methods Transgenic mice with sickle cell disease (SAD mice) and wild-type mice were exposed to an ischemic/reperfusion stress. The following parameters were evaluated: hematologic profile, transaminase and bilirubin levels, liver histopathology, and mRNA levels of nuclear factor-κB p65, endothelial nitric oxide synthase, inducible nitric oxide synthase, heme oxygenase-1 and phosphodiesterase-1, -2, -3, and -4 genes in hepatocytes obtained by laser-capture microdissection. Immunoblotting was used to analyze the expression of the following proteins: nuclear factor-κB p65 and phospho-nuclear factor-κB p65, heme oxygenase-1, biliverdin reductase, heat shock protein-70, heat shock protein-27 and peroxiredoxin-6. A subgroup of SAD mice was treated with the phosphodiesterase-4 inhibitor rolipram (30 mg/Kg/day by gavage) during the ischemic/reperfusion protocol. Results In SAD mice the ischemic/reperfusion stress induced liver damage compatible with sickle cell disease hepatopathy, which was associated with: (i) lack of hypoxia-induced nuclear factor-κB p65 activation; (ii) imbalance in the endothelial/inducible nitric oxide synthase response to ischemic/reperfusion stress; (iii) lack of hypoxia-induced increased expression of heme oxygenase-1/biliverdin reductase paralleled by a compensatory increased expression of heat shock proteins 70 and 27 and peroxiredoxin-6; and (iv) up-regulation of the phosphodiesterase-1, -2, -3, and -4 genes. In SAD mice the phosphodiesterase-4 inhibitor rolipram attenuated the ischemic/reperfusion-related microcirculatory dysfunction, reduced the inflammatory cell infiltration and induced the heme oxygenase-1/biliverdin reductase cytoprotective systems. Conclusions In SAD mice, sickle cell hepatopathy is associated with perturbed nuclear factor-κB p65 signaling with an imbalance of endothelial/inducible nitric oxide synthase levels, lack of heme oxygenase-1/biliverdin reductase expression and up-regulation of two novel cytoprotective systems: heat shock protein-27 and peroxiredoxin-6.

Heat-shock protein-27, -70 and peroxiredoxin–II show molecular chaperone function in sickle red cells: Evidence from transgenic sickle cell mouse model

Sickle cell disease (SCD) is an autosomal recessive genetic red cell disorder characterized by the production of a defective form of hemoglobin, hemoglobin‐S, that is worldwide‐distributed. The acute clinical manifestations of SCD are related to hemoglobin cyclic‐polymerization and to the generation of rigid, dense red blood cells (RBCs). We studied RBCs membrane proteome from human sickle RBCs, fractioned according to density compared to normal RBCs. 2‐DE followed by MS analysis was carried out. We identified 65 proteins differently expressed, divided into five major clusters according to their functions: (i) membrane‐cytoskeleton proteins; (ii) metabolic enzymes; (iii) ubiquitin‐proteasome‐system; (iv) flotillins; (v) chaperones. HSP27, HSP70 and peroxiredoxin‐II (Prx‐II) showed the most relevant changes. They were differently recruited to sickle RBCs membrane in response to in vitro hypoxia. Potential markers were then validated in a transgenic‐mouse model for SCD, the SAD mice, exposed to hypoxia mimicking acute SCD vaso‐occlusive‐crisis (VOCs); we found that HSP70 and HSP27 bound to RBCs membrane respectively after 12 h and 48 h of hypoxia, while Prx‐II membrane binding was modulated during hypoxia. Our data indicate that HSP27 and HSP70 play a novel role as RBCs membrane protein protectors and as possibly new markers of severity of RBCs membrane damage during acute VOCs.

Dietary ω-3 fatty acids protect against vasculopathy in a transgenic mouse model of sickle cell disease

The anemia of sickle cell disease is associated with a severe inflammatory vasculopathy and endothelial dysfunction, which leads to painful and life-threatening clinical complications. Growing evidence supports the anti-inflammatory properties of ω-3 fatty acids in clinical models of endothelial dysfunction. Promising but limited studies show potential therapeutic effects of ω-3 fatty acid supplementation in sickle cell disease. Here, we treated humanized healthy and sickle cell mice for 6 weeks with ω-3 fatty acid diet (fish-oil diet). We found that a ω-3 fatty acid diet: (i) normalizes red cell membrane ω-6/ω-3 ratio; (ii) reduces neutrophil count; (iii) decreases endothelial activation by targeting endothelin-1 and (iv) improves left ventricular outflow tract dimensions. In a hypoxia-reoxygenation model of acute vaso-occlusive crisis, a ω-3 fatty acid diet reduced systemic and local inflammation and protected against sickle cell-related end-organ injury. Using isolated aortas from sickle cell mice exposed to hypoxia-reoxygenation, we demonstrated a direct impact of a ω-3 fatty acid diet on vascular activation, inflammation, and anti-oxidant systems. Our data provide the rationale for ω-3 dietary supplementation as a therapeutic intervention to reduce vascular dysfunction in sickle cell disease.

The Interplay Between Peroxiredoxin-2 and Nuclear Factor-Erythroid 2 Is Important in Limiting Oxidative Mediated Dysfunction in β-Thalassemic Erythropoiesis

AIMS β-Thalassemia is a common inherited red cell disorder characterized by ineffective erythropoiesis and severe oxidative stress. Peroxiredoxin-2 (Prx2), a typical 2-cysteine peroxiredoxin, is upregulated during β-thalassemic erythropoiesis, but its contribution to stress erythropoiesis, a common feature of thalassemia, is yet to be fully defined. RESULTS Here, we showed that Prx2(-/-) mice displayed reactive oxygen species related abnormalities in erythropoiesis similar to that of Hbb(th3/+) mice associated with activation of redox response transcriptional factor nuclear factor-erythroid 2 (Nrf2). We generated β-thalassemic mice genetically lacking Prx2 (Prx2(-/-)Hbb(th3/+)) and documented a worsened β-thalassemic hematological phenotype with severe ineffective erythropoiesis. To further validate a key role of Prx2 in stress erythropoiesis, we administrated fused recombinant PEP1Prx2 to Hbb(th3/+) mice and documented a decrease in ineffective erythropoiesis. We further show that Prx2 effects are mediated by activation of Nrf2 and upregulation of genes that protect against oxidative damage such as gluthatione S-transferase, heme-oxygenase-1, and NADPH dehydrogenase quinone-1. INNOVATION We propose Prx2 as a key antioxidant system and Nrf2 activation is a cellular adaptive process in response to oxidative stress, resulting in upregulation of antioxidant (antioxidant responsive element) genes required to ensure cell survival. CONCLUSION Our data shed new light on adaptive mechanisms against oxidative damage through the interplay of Prx2 and Nrf2 during stress erythropoiesis and suggest new therapeutic options to decrease ineffective erythropoiesis by modulation of endogenous antioxidant systems.

Pharmacological inhibition of calpain-1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease

Sickle cell disease (SCD) is a globally distributed hereditary red blood cell (RBC) disorder. One of the hallmarks of SCD is the presence of circulating dense RBCs, which are important in SCD‐related clinical manifestations. In human dense sickle cells, we found reduced calpastatin activity and protein expression compared to either healthy RBCs or unfractionated sickle cells, suggesting an imbalance between activator and inhibitor of calpain‐1 in favor of activator in dense sickle cells. Calpain‐1 is a nonlysosomal cysteine proteinase that modulates multiple cell functions through the selective cleavage of proteins. To investigate the relevance of this observation in vivo, we evaluated the effects of the orally active inhibitor of calpain‐1, BDA‐410 (30 mg/kg/d), on RBCs from SAD mice, a mouse model for SCD. In SAD mice, BDA‐410 improved RBC morphology, reduced RBC density (D20; from 1106±0.001 to 1100±0.001 g/ml; P<0.05) and increased RBC‐K+ content (from 364±10 to 429±12.3 mmol/kg Hb; P<0.05), markedly reduced the activity of the Ca2+‐activated K+channel (Gardos channel), and decreased membrane association of peroxiredoxin‐2. The inhibitory effect of calphostin C, a specific inhibitor of protein kinase C (PKC), on the Gardos channel was eliminated after BDA‐410 treatment, which suggests that calpain‐1 inhibition affects the PKC‐dependent fraction of the Gardos channel. BDA‐410 prevented hypoxia‐induced RBC dehydration and K+ loss in SAD mice. These data suggest a potential role of BDA‐410 as a novel therapeutic agent for treatment of SCD.—De Franceschi, L., Franco, R. S., Bertoldi, M., Brugnara, C., Matté, A., Siciliano, A., Wieschhaus, A. J., Chishti, A. H., Joiner, C. H. Pharmacological inhibition of calpain‐1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease. FASEB J. 27, 750–759 (2013). www.fasebj.org